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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date; May 21 2018. Experimental completion date; May 24 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
Identification: FSM-005W
CAS Number: 2601-33-4
Batch: 170321
Purity: 19.9% aqueous solution of FSM-005W (solid)
Physical state/Appearance: clear colorless liquid
Expiry Date: 20 November 2018

Specific details on test material used for the study:
Name of the chemical substance (by IUPAC nomenclature): 1-Tetradecanaminium, N-(carboxymethyl)-N,N-dimethyl-,inner salt.
Alternative name: (abbr.: FSM-0005W)
CAS RN: -
Molecular Weight: -
Purity (5) of the chemical substance subjected to the study: 19.9%
Lot number of the chemical substance subjected to the study: 170321
Name and content of impurities: 2-proponal: 0.4 %. water 79.7%
Vapor pressure: -
Solubility in water: Easy soluble
1-octanol/water partition coefficient: -
Melting point: -
Boiling: -
Appearance at room temperature: Colourless/ Liquid
Stability: -
Storage condition: refrigeration, shaded, air tight

After the exposure period, the infrared absorptiuon spectrum of the test substance was measured. The spectrum obtained was consistent with that measured before the study, indication that the test substance was stable under the storage conditions.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentrations of the test substance in all test solutions and test cultures were measured at the beginning of the exposure and at the 24m 48 and 72 hours after the beginning of the exposure by Liquid Chromatography/Mass Spectrometry (LC/MS).

Test solutions

Vehicle:
yes
Remarks:
Dilution water
Details on test solutions:
Preparation of stock solution
Test substance: 20 mg
Purity of test substance: 19.9 %
Dilution water: OECD medium 22 °C
Final volume: 200 mL
Vessel: Mess bottle
Concentration: 19.9 mg/L (concentration corrected by the purity)
Mixing method: Shake by hand

Preparation of test soluiton for pre-conditioning
Stock solution is collected as shown below and diluted with the medium.
The test solution for control control is the medium
Final Volume: 100 mL
Vessel: 300 mL Erlenmeyer flask with aluminium cap
Mixing method: shake by hand
Seperation factor: 1.78
Replicates: 6 vessels/control group, 3 vessels/concentration group
1 extra vessel/test group,
4 vessels for collection analysis sample/test group

Nominal concentration mg/L* Group Name (abbreviation) stock Sol. mL
Control C 0
0.050 Conc 1 0.251
0.089 Conc 2 0.447
0.16 Conc 3 0.804
0.28 Conc 4 1.413
0.50 Conc 5 2.51
*Concentration corrected for the purity of the substance.

Shaking method for pre-conditioning: 100 rpm
Duration for pre-conditioning: 72 hours

Preparation of test solution
Discard the pre-conditioning test solution 72 hours later and prepare the test solution in the same manner as shown above.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test Organisms
1) Common name: Unicellular green algae
2) Scientific name: Pseudokirchneriella subcapitata
3) Strain: ATCC22662
4) Source: American Type Culture Collection
5) Date of receipt: June 20, 1996
6) Maintenance of obtained algae:
Algae have been maintained by period subculture using Gorham medium, Algae are kept under sterile condition. Sterile test has been performed periodically, every six months, to confirm that the algae are not contaminated.
7) Sensitivity to a reference substance:
Growth inhibition test of a reference substance (potassium dichromate, guaranteed reagent) has been conducted periodically (approximately every six months) and 72-hour medium growth inhibition concentrations were estimated by comparison of growth rates (ErC50). The latest value ofErC50 was follows.
ErC50 = 0.710 mg/L (95% confidence limits: 0.677-0.746 mg/L, Exposure period: From June 19 to 22, 2018)
The average values of ErC50 since June, 2000 were as follows.
Average ErC50 ± standard deviation = 0.807 ± 0.0704 mg/L, n=37, (Minimum-maximum = 0.687-0.965 mg/L)
8) Conditions of preculture:
Preculture period: From May 18 to 21, 2018
In order to adapt the algae to the test conditions and ensure that the algae were in the exponential growth phase when used to inoculate the test solutions, the algae were precultured under the same conditions as the test culture. Neither modified nor unusual cells were confirmed.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22 °C, variation range over the exposure period is ±2 °C
pH:
Not adjusted
7.6-7.7 at 0 hours and 7.8-8.0 at 72 hours
Nominal and measured concentrations:
Range finding test:
Nominal concentrations: 0.020, 0.050, 0.50, 1.0

Main test
Nominal Concentrations: 0.050, 0.089, 0.16, 0.28, 0.50
Measured Concentration: 0.0531, 0.0957, 0.315, 0.548 mg/L
Details on test conditions:
1) Beginning of exposure
A certain quantity of the inoculum culture was added to the test vessels, so that the initial biomass based cell concentration as surrogate parameter, in the test solution was 5 X 10^3 cells/mL. Biomass in the inoculum culture was determined with an electronic particle counter as well as microscopically using a hemacytometer. Algae were not inoculated in the extra vessels and the vessels for collecting analysis sample at the beginning of exposure.
The exposure period started by placing the test vessels in the culturing apparatus according to the table of random sampling digits. These vessels were re-arranged every 24 hours. Although the extra vessels and the vessels for collecting analysis sample were also placed in the culturing apparatus, the vessels for collecting analysis sample at the beginning of exposure was not placed.

2) Measurement of biomass
The biomass of algae in each test vessel was measured at 24, 48 and 72 hours after the beginning of exposure. One milliliter oftest culture (0.5 mL at 72 hours) was suspended in 9.0 mL of electrolyte (9.5 mL at 72 hours) to count the algal cells by electronic particle counter.

3) Color observation of test culture and microscopic observation of algae
The color of the test cultures was observed by comparison with the extra vessel of each test group at the beginning of exposure and at 24, 48 and 72 hours after the beginning of exposure. At 72 hours after the beginning of exposure, appearance of algae was observed through the microscope.

4) Measurement of test conditions
The pH values of the test solutions and the test cultures were measured at the beginning of exposure and at 72 hours after the beginning of exposure. At the beginning of exposure, a part of the test solution in the extra vessel was collected and pH was measured. At 72 hours after the beginning of exposure, pH of the test culture was measured in one vessel (No. 1) in each test group. The pH of Conc. 3 was measured in all test vessels, since the biomass differed more than twice in the same test group. During the exposure period, temperatures, light intensities, and revolutions in the culturing apparatus were measured at least once a day.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.173 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.053 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Conditions in the Culturing Apparatus, pH of Test Solutions and Test Cultures, and Appearance of Test Solutions
The temperature in the culturing apparatus during exposure period ranged within 22 ± 2 °C, nominal value. The pH value of test solutions at the beginning of exposure were 7.6 to 7.7 and test cultures at 72 hours after the beginning of exposure were 7.8 to 8.0.
Test solutions before inoculation were colorless, and showed no suspended solids, no floating solids, and no precipitates in all test groups.

50% Growth Inhibition Concentration (EC50) and No Observed Effect Concentration (NOEC)
It was confirmed that algae grew exponentially in the control group.
The ErC50 and NOECr values are shown below.
50% growth inhibition concentration, ErC50 (0-72h): 0.173 mg/L (95% confidence limits: 0.154—0.194 mg/L, least squares linear regression analysis)
No observed effect concentration, NOECr (0-72h): 0.0531 mg/L
The NOECr value was determined by an analysis of variance (ANOVA), Williams test, subsequent to Bartlett test for homogeneity of variances. The significance level for all tests was set at 0=0.05, except Bartlett test, which was set at 0=0.01.

Observation of Algae
In the control group and Conc. 1 to 2, the color of test cultures observed with naked eye showed a tendency to get more greenish during the exposure period. In 1 vessel of Conc. 3, the color of test cultures was slightly changed, but showed no tendency to get greenish, whereas in other vessels of Conc.3, the color of test cultures observed with naked eye showed a tendency to get more greenish during the exposure period. In Conc. 4 and 5, the color of test cultures was slightly changed, but showed no tendency to get greenish.
By microscopic observation at the end of exposure, in Conc.l and 2 neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with that in the control group. The cell volume expansion was partially observed in Conc. 3, 4 and 5.

Validity of the Test
The biomass in the control cultures increased exponentially above 1 6 fold during 72-hour culture. The mean coefficient of variation for section by section specific growth rates in the control cultures and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 35% and 7%, respectively. This test is valid because these results met the test validity criteria.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The results were calculated from the time-weighted mean of measured concentrations.
50% growth inhibition concentration, ErC50 (0-72h): 0.173 mg/L (95% confidence limits: 0.154—0.194 mg/L, least squares linear regression analysis)
No observed effect concentration, NOECr (0-7211): 0.0531 mg/L
Executive summary:

Methods

The study was conducted in accordance with the OECD Guidelines for the Testing of Chemicals No.201 "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (2006).

The test was performed with correction of the concentration by the purity (19.9%) of test substance. There was a concern that the test substance will be adsorbed to the test vessel. Therefore, pre-conditioning was performed using the test solution and then the exposure test was conducted.

 1) Test Organisms:  Pseudokirchneriella subcapitata, Unicellular green algae

 2) Dilution Water: The medium recommended by the Test Guideline

3) Duration: 72 hours

4) Exposure procedure: Static, open system and skaing (100 rpm)

5) Initial biomass: 5 x 103cells/mL, algae of exponential growth phase (dried weight of algae at the exponential growth phase: 1.8 x 10^-8 mg/cell, n=26)

6) Temperature: 22°C, variation range over the exposure period is within ±2 °C

7) 65 to 75 yE/m2/s, continuous illumination with white fluorescent lamp (on the surface of test culture)

8) Test concentration:

Test group

Nominal Concentration

(mg/L)

 Control  
 Conc 1

 0.050

Conc 2   0.089
 Conc 3 0.16 
Conc 4   0.28
 Conc 5  0.50

Separation factor: 1.8

* : Concentration corrected by the purity of the test substance

9) Analytical methods: Liquid chromatography/mass spectrometry (LC/MS)

Results

1) Concentrations of test substance in test solutions and test cultures

The time-weighted means of measured concentrations of the test substance in Conc. 1 to 5were 0.0531, 0.0957, 0.179, 0.315 and 0.548 mg/L, respectively.

2) Inhibition concentration by comparison of growth rates

The results were calculated from the time-weighted mean of measured concentrations.

50% growth inhibition concentration, ErC50 (0-72h): 0.173 mg/L (95% confidence limits: 0.154—0.194 mg/L, least squares linear regression analysis)

No observed effect concentration, NOECr (0-7211): 0.0531 mg/L

3) Observation of the appearance of algae

By microscopic observation at the end of exposure, in Conc. I and 2 neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, andthe algae looked normal compared with that in the control group. The cell volume expansion was partially observed in Conc. 3, 4 and 5.